Development of genomic resources in the domestic cat is key to application of this species as a model for heritable and infectious diseases, and for comparative genome analysis. Previous versions of the cat gene map, based on somatic cell hybrid analysis, revealed considerable conservation of synteny with the human genome. However, these maps provided no knowledge of gene order and genome rearrangement between the two species, information which is critical to applying the comparative candidate approach to positional cloning in gene-poor model systems. Radiation hybrid (RH) mapping has emerged as the most powerful tool for constructing moderate- to high-density gene maps in vertebrates by obviating the need to identify interspecific polymorphisms. A domestic cat RH panel has been developed and serves as the backbone for feline gene mapping, while also serving to cross-reference microsatellite positions on the current domestic cat-Asian leopard cat interspecies linkage map. We have completed a third generation 1126-locus map which adds 301 polymorphic microsatellite loci, more than doubling microsatellite density in a previous RH map. The third generation integrates 572 coding loci (expressed sequence tags, genes, and comparative anchor tagged sequences CATS markers) with 554 microsatellites. Microsatellites now span every autosome with an average spacing of roughly one polymorphic short tandem repeat (STR) every 5 centimorgans, and a total marker resolution (Type I and Type II loci) of one locus every 2.7 megabases. The current feline radiation hybrid map now incorporates sufficient Type I and Type II markers to facilitate the identification of genes, using comparative candidate positional clone analysis, controlling phenotypes of interest such as those relating to hereditary disease, coat color, patterning and morphology. Over 250 hereditary phenotypes have been described in the domestic cat. These resources can also be extended to the remaining 36 species of the cat family for population genetic and evolutionary genomic analyses. We are currently increasing the Type I marker density to nearly 1000 loci through additional expressed sequence tag (EST) development and bacterial artificial chromosome (BAC)-end sequencing-based marker production, focusing primarily on gaps in human-cat synteny. Presently we are also increasing microsatellite coverage at least five-fold with an ultimate goal of 1000-1500 microsatellites on the map. Further refinement of rearrangement breakpoints and conserved segment boundaries will enhance cross-reference to other mammalian species' genomes for use in diverse studies of genetics and genome evolution.